Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Protocol: ATCCNO: 11745 SPEC: See general instructions for thawing and storage of frozen material before proceeding. Add thawed contents to a single 16 x 125 mm glass screw-capped test tube of the appropriate medium. Incubate the culture vertically with the cap screwed on tightly. It is essential to establish cultures initially in small volumes. Once established, the culture can be scaled up to larger volumes. Vigorously agitate the culture and aseptically transfer 0.1 ml of culture to a fresh tube of medium weekly.

Subcultivation

Protocol: ATCCNO: 11745 SPEC: See general instructions for thawing and storage of frozen material before proceeding. Add thawed contents to a single 16 x 125 mm glass screw-capped test tube of the appropriate medium. Incubate the culture vertically with the cap screwed on tightly. It is essential to establish cultures initially in small volumes. Once established, the culture can be scaled up to larger volumes. Vigorously agitate the culture and aseptically transfer 0.1 ml of culture to a fresh tube of medium weekly.

Cryopreservation

1. Harvest cells from cultures that are at or near peak density. Aseptically transfer the broth overlay to a plastic centrifuge tube and adjust the concentration of cells to 2 x 107/ml in fresh medium (broth overlay). If necessary, cells may be concentrated by centrifugation at 800 x g for 5 min.

3. Mix the cell preparation and the DMSO solution in equal portions. The final concentration will be 107 cells/ml and 5% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO solution to the start of the freezing process should be no less than 15 min and no longer than 30 min.

5. Place the vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If the freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through the heat of fusion. At -40°C plunge into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus. Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen. (The cooling rate in this apparatus is approximately

-1°C/min.)

6. The frozen preparations should be stored in either the vapor or liquid phase of a nitrogen refrigerator. Frozen preparations stored below -130°C are stabile indefinitely. Those stored at temperatures above -130°C are progressively less stabile as the storage temperature is elevated. Vials should not be stored above -70°C.

7. To establish a culture from the frozen state place an ampule in a water bath set at 35°C. Immerse the vial just to a level just above the surface of the frozen material. Do not agitate the vial.

8. Immediately after thawing, do not leave in the water bath, aseptically remove the contents of the ampule and inoculate a 16 x 125 mm screw-capped test tube containing ATCC medium 807.

9. Incubate the culture vertically at 25°C. Observe the culture daily and transfer when numerous trophozoites are observed.

Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.